The long term objective of this proposal is to elucidate the processes involved in transmitter release, and in particular the involvement of synaptic vesicles. To achieve this goal, it is proposed to use a preparation, the Drosophila mutant, shibire-ts, in which vesicle recycling is reversibly blocked at 29 degrees C. This allows the experimenter to precisely -control the number of vesicles in the synapse by evoking transmitter release at 29 degrees C, gradually depleting the synapse to the desired level. Also, following complete depletion at 29 degrees C, a low temperature pulse at 19 degrees C allows a limited amount of vesicle recycling to occur, the number of vesicles reformed being dependent on the duration of the pulse. This situation allows a straightforward analysis of synaptic depression, which is normally complicated by simultaneously occurring vesicle recycling at an unknown rate. From depression plots from shi at 29 degrees C, available quanta (n) can be determined precisely and correlated with a morphological structure. These plots demonstrate a 2-phase decay, apparently related to readily-releasable and reserve populations, which have two different probabilities of release. The characteristics of these two populations will be investigated using physiological, morphological and immunocytochemical techniques. The involvement of Ca++ and Mg++, and of synapsin I in the transition from reserve to readily-releasable status win be investigated. By allowing a limited amount of vesicle reformation after depletion to occur, a population composed solely of the readily- releasable variety can be created. Thus, the two populations can be investigated separately. If Ca++ is involved in more than one process, e.g., transition from reserve to releasable states and fusion, these can be distinguished. The involvement of synaptophysin and synapsin I will be investigated by using anti-synaptophysin antibody staining, and by injection of antibodies against these proteins into the presynaptic terminal. Also, synaptophysin antibody staining will be used to determine the fate of vesicle membrane after exocytosis and during the recycling process. Electron microscopy of serially-sectioned synapses will be performed after all physiological experiments and a morphological correlate for the two populations will be sought. This research will provide an understanding of neural processes which can be basic for the further understanding and treatment of many neurological diseases.